In this study, variation of beta-glucan content was assessed in 20 barley line and cultivars based on complete block design with three replications. Genetic diversity of these genotypes was also evaluated using ISSR markers. Beta-glucan extracted by an enzymatic method. Significant differences were found at the level of 1% among barley genotypes for beta-glucan content. The beta glucan content was variable from 7.21 to 12.48 and, the Yosef, E94B3 and E94B17 genotypes hold the highest content of the beta-glucan. ISSR primers with average polymorphism of 66.79%, genetic diversity of 0.25 and Shannon index of 0.37 were determined as efficient markers for studying genetic diversity. The barley lines and cultivars were assigned in two distinct groups according to their genetic pedigree. On the basis of non-parametric Kruskal-Wallis, Spearman correlation, and stepwise regression analysis, nine informative primers were detected explaining highest seed’s beta-glucan content variations ranging from 24.3 to 42.4 percent. The ISSR6 (700), the combination of ISSR1+ISSR4 (1400) and IS2+ ISSR2 (1400) primers were the most informative primers for the beta-glucan content. The informative markers provide possible functional and efficient marker based selection method and, screening the barley germplasms for the highest beta-glucan content.